As a next-generation DNA sequencer, a method of electrically measuring the base sequence of DNA directly without performing extension reactions or fluorescence labeling has been drawing attention. To that end, a nanopore DNA sequencing method that determines a base sequence by directly measuring a DNA fragment without using a reagent has been actively researched and developed. This method is based on the principle of sequentially identifying the types of individual bases contained in a DNA strand by directly measuring the difference between the types of the bases on the basis of the amounts of blocking currents that flow when the DNA strand passes through a nanopore. Such a method is expected to increase the throughout, reduce the running cost, and be able to determine long sequences of bases since amplification using enzymes of template DNA is not performed, nor is a labeling substance such as a phosphor used.
As a problem of the nanopore method, translocation control for DNA that passes through a nanopore is given. In order to measure the difference between the types of individual bases contained in a DNA strand on the basis of the amounts of blocking currents, it is considered that the speed of the DNA passing through a nanopore should be set to greater than or equal to 100 μs per 1 base in view of current noise generated during measurement and a time constant of fluctuation of DNA molecules. When DNA is sequenced using a nanopore, a potential gradient is formed using electrodes located above and below the nanopore so that negatively charged DNA is allowed to pass through the nanopore. However, the speed of DNA passing through a nanopore is typically as high as less than or equal to 1 μs per base, and it is thus difficult to sufficiently measure a blocking current derived from each base.
As a translocation control method, there is known a method that includes immobilizing an end of the target DNA to be read on an end of a probe and controlling a minute displacement of the probe using an external drive mechanism (a motor and a piezoelectric element), thereby controlling the movement of the DNA passing through a nanopore. In Non Patent Literature 1 and 2, DNA is immobilized on an end of a probe of an atomic force microscope (AFM) so that the DNA is introduced into a nanopore. DNA is negatively charged in an aqueous solution. Therefore, it is introduced into a nanopore by receiving a force due to a potential difference generated around the nanopore. Herein, since an atomic force microscope is used, DNA is immobilized on an AFM probe. Therefore, a phenomenon that the DNA receives an attractive force from an electric field around the nanopore can be monitored from deflection of the probe that occurs when the AFM probe is pulled by the DNA. At the same time, monitoring an ion current that vertically flows through the nanopore can acquire blocking signals generated when the DNA passes through the nanopore. Since it was confirmed that such signals are synchronized with one another, it was verified that both dsDNA and ssDNA can be introduced into and extracted from the nanopore.